Professor of Civil and Environmental Engineering, of Chemical Engineering and, by courtesy, of Biology

Bio

In our research we investigate molecular microbial metabolism and its linkage to ecological and evolutionary processes. We explore the distinguishing features of novel microbial metabolism and how molecular and biochemical differences in metabolism shape microbial fitness. We study novel microbial metabolism with relevance to bioremediation, bioenergy, and intestinal microbiology.

1) Microbial Reductive Dehalogenation

Chloroethenes, such as PCE and TCE, are the most prevalent groundwater contaminants in the U.S. and the developed countries. Large scale remediation of contaminated aquifers relies largely on the activity of a group of unusual microbes (Dehalococcoides) that derive energy from reductive dehalogenation. We study reductive dehalogenases and the strictly anaerobic bacteria, such as Dehalococcoides mccartyj and Shewanella, on a biochemical, physiological, genomic, and population level to better understand the unprecedented biochemistry of the coenzyme B 12-containing reductive dehalogenases. We also use this information to improve chlorethene bioremediation. Population-level studies in our lab have been revealing speciation and niche adaptation in Dehalococcoides mccartyj. in response to subtle changes in physical-chemical environments.

2) Microbial Electrosynthesis and Electron Transport between Microbes and Surfaces

Some microbes have the the capacity to either derive metabolic electrons from redox-active mineral surfaces or transfer such electrons to these surfaces. These processes are of great relevance to geochemical, environmental, but also bioenergy processes. We are investigating the molecular bases of such novel electron transfer to uncover the enzymes and pathways for electron uptake. More recently, we began to explore microbial electrosynthesis as a novel means to produce CO2-neutral biofuels and commodity chemicals.

3) Microbial Metabolic Processes in the Large Intestine

Irritable Bowel Syndrome (IBS) is a chronic, episodic gastrointestinal disorder that is characterized by abdominal pain and altered bowel habits. IBS prevalence is estimated to be 10-15% in Western countries comprising 25 to 50 percent of all referrals to gastroenterologists. The gastrointestinal tract harbors a complex and diverse microbial community, which plays important roles in host nutrition, immune function, health and disease, and it is hypothesized the IBS disease phenotype is associated with a change in colonic microbiota and/or host factors such as mucosal function and immunity. With our physician collaborator, we study the metabolic processes in the intestinal microbial community, and how cellular metabolism is controlled by the host mucosa.

Abstract

Past studies of hydrogen cycling in hypersaline microbial mats have shown an active nighttime cycle, with production largely from Cyanobacteria and consumption from sulfate-reducing bacteria (SRB). However, the mechanisms and magnitude of hydrogen cycling have not been extensively studied. Two mats types near Guerrero Negro, Mexico-permanently submerged Microcoleus microbial mat (GN-S), and intertidal Lyngbya microbial mat (GN-I)-were used in microcosm diel manipulation experiments with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), molybdate, ammonium addition, and physical disruption to understand the processes responsible for hydrogen cycling between mat microbes. Across microcosms, H2 production occurred under dark anoxic conditions with simultaneous production of a suite of organic acids. H2 production was not significantly affected by inhibition of nitrogen fixation, but rather appears to result from constitutive fermentation of photosynthetic storage products by oxygenic phototrophs. Comparison to accumulated glycogen and to CO2 flux indicated that, in the GN-I mat, fermentation released almost all of the carbon fixed via photosynthesis during the preceding day, primarily as organic acids. Across mats, although oxygenic and anoxygenic phototrophs were detected, cyanobacterial [NiFe]-hydrogenase transcripts predominated. Molybdate inhibition experiments indicated that SRBs from a wide distribution of DsrA phylotypes were responsible for H2 consumption. Incubation with (13)C-acetate and NanoSIMS (secondary ion mass-spectrometry) indicated higher uptake in both Chloroflexi and SRBs relative to other filamentous bacteria. These manipulations and diel incubations confirm that Cyanobacteria were the main fermenters in Guerrero Negro mats and that the net flux of nighttime fermentation byproducts (not only hydrogen) was largely regulated by the interplay between Cyanobacteria, SRBs, and Chloroflexi.

Abstract

Mercury sulfides (cinnabar and metacinnabar) are the main ores of Hg and are relatively stable under oxic conditions (Ksp = 10⁻⁵⁴ and 10⁻⁵² , respectively). However, until now their stability in the presence of micro-organisms inhabiting acid mine drainage (AMD) systems was unknown. We tested the effects of the AMD microbial community from the inoperative Hg mine at New Idria, CA, present in sediments of an AMD settling pond adjacent to the main waste pile and in a microbial biofilm on the surface of this pond, on the solubility of crystalline HgS. A 16S rRNA gene clone library revealed that the AMD microbial community was dominated by Fe-oxidizing (orders Ferritrophicales and Gallionellas) and S-oxidizing bacteria (Thiomonas sp.), with smaller amounts (≤ 6%) being comprised of the orders Xanthomondales and Rhodospirillales. Though the order Ferritrophicales dominate the 16S rRNA clones (>60%), qPCR results of the microbial community indicate that the Thiomonas sp. represents ~55% of the total micro-organisms in the top 1 cm of the AMD microbial community. Although supersaturated with respect to cinnabar and metacinnabar, microcosms inoculated with the AMD microbial community were capable of releasing significantly more Hg into solution compared to inactivated or abiotic controls. Four different Hg-containing materials were tested for bacterially enhanced HgS dissolution: pure cinnabar, pure metacinnabar, mine tailings, and calcine material (processed ore). In the microcosm with metacinnabar, the presence of the AMD microbial community resulted in an increase of dissolved Hg concentrations up to 500 μg L ⁻¹during the first 30 days of incubation. In abiotic control microcosms, dissolved Hg concentrations did not increase above 100 ng L⁻¹ . When Hg concentrations were below 50 μg L⁻¹ , the Fe-oxidizing bacteria in the AMD microbial community were still capable of oxidizing Fe(II) to Fe(III) in the AMD solution, whereas concentrations above 50 μg L⁻¹ resulted in inhibition of microbial iron oxidation. Our experiments show that the AMD microbial community contributes to the dissolution of mercury sulfide minerals. These findings have major implications for risk assessment and future management of inoperative Hg mines worldwide.

Abstract

Defining the functional status of host-associated microbial ecosystems has proven challenging owing to the vast number of predicted genes within the microbiome and relatively poor understanding of community dynamics and community-host interaction. Metabolomic approaches, in which a large number of small molecule metabolites can be defined in a biological sample, offer a promising avenue to 'fingerprint' microbiota functional status. Here, we examined the effects of the human gut microbiota on the fecal and urinary metabolome of a humanized (HUM) mouse using an optimized ultra performance liquid chromatography-mass spectrometry-based method. Differences between HUM and conventional mouse urine and fecal metabolomic profiles support host-specific aspects of the microbiota's metabolomic contribution, consistent with distinct microbial compositions. Comparison of microbiota composition and metabolome of mice humanized with different human donors revealed that the vast majority of metabolomic features observed in donor samples are produced in the corresponding HUM mice, and individual-specific features suggest 'personalized' aspects of functionality can be reconstituted in mice. Feeding the mice a defined, custom diet resulted in modification of the metabolite signatures, illustrating that host diet provides an avenue for altering gut microbiota functionality, which in turn can be monitored via metabolomics. Using a defined model microbiota consisting of one or two species, we show that simplified communities can drive major changes in the host metabolomic profile. Our results demonstrate that metabolomics constitutes a powerful avenue for functional characterization of the intestinal microbiota and its interaction with the host.The ISME Journal advance online publication, 6 June 2013; doi:10.1038/ismej.2013.89.

Abstract

Shewanella oneidensis MR-1, a gammaproteobacterium with respiratory versatility, forms biofilms on mineral surfaces through a process controlled by the cyclic dinucleotide messenger c-di-GMP. Cellular concentrations of c-di-GMP are maintained by proteins containing GGDEF and EAL domains, which encode diguanylate cyclases for c-di-GMP synthesis and phosphodiesterases for c-di-GMP hydrolysis, respectively. The S. oneidensis MR-1 genome encodes several GGDEF and EAL domain proteins (50 and 31, respectively), with a significant fraction (∼10) predicted to be multidomain (e.g., GGDEF-EAL) enzymes containing an additional Per-Arnt-Sim (PAS) sensor domain. However, the biochemical activities and physiological functions of these multidomain enzymes remain largely unknown. Here, we present genetic and biochemical analyses of a predicted PAS-GGDEF-EAL domain-containing protein, SO0437, here named PdeB. A pdeB deletion mutant exhibited decreased swimming motility and increased biofilm formation under rich growth medium conditions, which was consistent with an increase in intracellular c-di-GMP. A mutation inactivating the EAL domain also produced similar swimming and biofilm phenotypes, indicating that the increase in c-di-GMP was likely due to a loss in phosphodiesterase activity. Therefore, we also examined the enzymatic activity of purified PdeB and found that the protein exhibited phosphodiesterase activity via the EAL domain. No diguanylate cyclase activity was observed. In addition to the motility and biofilm phenotypes, transcriptional profiling by DNA microarray analysis of biofilms of pdeB (in-frame deletion and EAL) mutant cells revealed that expression of genes involved in sulfate uptake and assimilation were repressed. Addition of sulfate to the growth medium resulted in significantly less motile pdeB mutants. Together, these results indicate a link between c-di-GMP metabolism, S. oneidensis MR-1 biofilm development, and sulfate uptake/assimilation.

Abstract

S. oneidensis MR-1 is a dissimilatory metal-reducing bacterium. Under anoxic conditions S. oneidensis MR-1 attaches to and uses insoluble minerals such as Fe(III) and Mn(IV) oxides as electron acceptors. In the laboratory, S. oneidensis MR-1 forms biofilms under hydrodynamic flow conditions on a borosilicate glass surface; formation of biofilms was previously found to be dependent on the mxd gene cluster (mxdABCD).This study revealed environmental and genetic factors regulating expression of the mxd genes in S. oneidensis MR-1. Physiological experiments conducted with a S. oneidensis MR-1 strain carrying a transcriptional lacZ fusion to the mxd promoter identified electron donor starvation as a key factor inducing mxd gene expression. Tn5 mutagenesis identified the ArcS/ArcA two-component signaling system as a repressor of mxd expression in S. oneidensis MR-1 under planktonic conditions. Biofilms of ∆arcS and ∆arcA strains carrying a transcriptional gfp -reporter fused to the mxd promoter revealed a reduced mxd expression, suggesting that ArcS/ArcA are necessary for activation of mxd expression under biofilm conditions. Biofilms of ∆arcS and ∆arcA mutants were unable to form a compact three-dimensional structure consistent with a low level of mxd expression. In addition, BarA/UvrY was identified as a major regulator of mxd expression under planktonic conditions. Interestingly, biofilms of ∆barA and ∆uvrY mutants were able to form three-dimensional structures that were, however, less compact compared to wild type biofilms.We have shown here that the mxd genes in S. oneidensis MR-1 are controlled transcriptionally in response to carbon starvation and by the ArcS/ArcA and the BarA/UvrY signaling system. BarA might function as a sensor to assess the metabolic state of the cell, including carbon starvation, leading to expression of the mxd operon and therefore control biofilm formation.

Abstract

The genome sequence of psychrophilic Shewanella sediminis revealed the presence of five putative reductive dehalogenases (Rdhs). We found that cell extracts of pyruvate/fumarate-grown S. sediminis cells catalysed reduced methyl viologen-dependent reductive dechlorination of tetrachloroethene (PCE) to trichloroethene (TCE) at a specific activity of approximately 1 nmol TCE min(-1) (mg protein)(-1). Dechlorination of PCE followed Michaelis-Menten kinetics with an apparent Km of 120 μM PCE. No PCE dechlorination was observed with heat-denatured extract or when cyanocobalamin was omitted from the growth medium; however, the presence of PCE in the growth medium increased PCE transformation rates. Analysis of mutants carrying in-frame deletions of all five Rdhs encoding genes showed that only deletion of Ssed_3769 resulted in the loss of PCE dechlorination activity suggesting that Ssed_3769 is a functional Rdh. This is the first study to show reductive dechlorination activity of PCE in a sediment-dwelling Shewanella species that may be important for linking the flux of organohalogens to organic carbon via reductive dehalogenation in marine sediments.

Abstract

Photosynthetic microbial mats possess extraordinary phylogenetic and functional diversity that makes linking specific pathways with individual microbial populations a daunting task. Close metabolic and spatial relationships between Cyanobacteria and Chloroflexi have previously been observed in diverse microbial mats. Here, we report that an expressed metabolic pathway for the anoxic catabolism of photosynthate involving Cyanobacteria and Chloroflexi in microbial mats can be reconstructed through metatranscriptomic sequencing of mats collected at Elkhorn Slough, Monterey Bay, CA, USA. In this reconstruction, Microcoleus spp., the most abundant cyanobacterial group in the mats, ferment photosynthate to organic acids, CO2 and H2 through multiple pathways, and an uncultivated lineage of the Chloroflexi take up these organic acids to store carbon as polyhydroxyalkanoates. The metabolic reconstruction is consistent with metabolite measurements and single cell microbial imaging with fluorescence in situ hybridization and NanoSIMS.

Abstract

Results are presented from a chemostat study where the reductive dehalogenation of PCE was evaluated in the absence and presence of sulfate. Two chemostats inoculated with the Point Mugu culture, which contains strains of Dehalococcoides mccartyi, were operated at a 50 day HRT and fed PCE (1.12 mM) and lactate (4.3 mM). The control chemostat (PM-5L, no sulfate), achieved pseudo-steady-state transformation of PCE to ethene (98%) and VC (2%) at 2.4 nM of H(2). Batch kinetic tests with chemostat harvested cells showed the maximum rate (k(max)X) value for each dehalogenation step remained fairly constant, while hupL clone library analyses showed maintenance of a diverse D. mccartyi community. Sulfate (1 mM) was introduced to the second chemostat, PM-2L. Effective sulfate reduction was achieved 110 days later, resulting in 600 μM of total sulfide. PCE dechlorination efficiency decreased following complete sulfate reduction, yielding ethene (25%), VC (67%), and cis-DCE (8%). VC dechlorination was most affected, with k(max)X values decreasing by a factor of 50. The decrease was associated with the enrichment of the Cornell group of D. mccartyi and decline of the Pinellas group. Long-term exposure to sulfides and/or competition for H(2) may have been responsible for the community shift.

Abstract

We determined a significant fraction of the genome sequence of a representative of Thiovulum, the uncultivated genus of colorless sulfur Epsilonproteobacteria, by analyzing the genome sequences of four individual cells collected from phototrophic mats from Elkhorn Slough, California. These cells were isolated utilizing a microfluidic laser-tweezing system, and their genomes were amplified by multiple-displacement amplification prior to sequencing. Thiovulum is a gradient bacterium found at oxic-anoxic marine interfaces and noted for its distinctive morphology and rapid swimming motility. The genomic sequences of the four individual cells were assembled into a composite genome consisting of 221 contigs covering 2.083 Mb including 2,162 genes. This single-cell genome represents a genomic view of the physiological capabilities of isolated Thiovulum cells. Thiovulum is the second-fastest bacterium ever observed, swimming at 615 ?m/s, and this genome shows that this rapid swimming motility is a result of a standard flagellar machinery that has been extensively characterized in other bacteria. This suggests that standard flagella are capable of propelling bacterial cells at speeds much faster than typically thought. Analysis of the genome suggests that naturally occurring Thiovulum populations are more diverse than previously recognized and that studies performed in the past probably address a wide range of unrecognized genotypic and phenotypic diversities of Thiovulum. The genome presented in this article provides a basis for future isolation-independent studies of Thiovulum, where single-cell and metagenomic tools can be used to differentiate between different Thiovulum genotypes.

Abstract

N(2) fixation is a key process in photosynthetic microbial mats to support the nitrogen demands associated with primary production. Despite its importance, groups that actively fix N(2) and contribute to the input of organic N in these ecosystems still remain largely unclear. To investigate the active diazotrophic community in microbial mats from the Elkhorn Slough estuary, Monterey Bay, CA, USA, we conducted an extensive combined approach, including biogeochemical, molecular and high-resolution secondary ion mass spectrometry (NanoSIMS) analyses. Detailed analysis of dinitrogenase reductase (nifH) transcript clone libraries from mat samples that fixed N(2) at night indicated that cyanobacterial nifH transcripts were abundant and formed a novel monophyletic lineage. Independent NanoSIMS analysis of (15)N(2)-incubated samples revealed significant incorporation of (15)N into small, non-heterocystous cyanobacterial filaments. Mat-derived enrichment cultures yielded a unicyanobacterial culture with similar filaments (named Elkhorn Slough Filamentous Cyanobacterium-1 (ESFC-1)) that contained nifH gene sequences grouping with the novel cyanobacterial lineage identified in the transcript clone libraries, displaying up to 100% amino-acid sequence identity. The 16S rRNA gene sequence recovered from this enrichment allowed for the identification of related sequences from Elkhorn Slough mats and revealed great sequence diversity in this cluster. Furthermore, by combining (15)N(2) tracer experiments, fluorescence in situ hybridization and NanoSIMS, in situ N(2) fixation activity by the novel ESFC-1 group was demonstrated, suggesting that this group may be the most active cyanobacterial diazotroph in the Elkhorn Slough mat. Pyrotag sequences affiliated with ESFC-1 were recovered from mat samples throughout 2009, demonstrating the prevalence of this group. This work illustrates that combining standard and single-cell analyses can link phylogeny and function to identify previously unknown key functional groups in complex ecosystems.

Abstract

Hydrogen (H(2)) release from photosynthetic microbial mats has contributed to the chemical evolution of Earth and could potentially be a source of renewable H(2) in the future. However, the taxonomy of H(2)-producing microorganisms (hydrogenogens) in these mats has not been previously determined. With combined biogeochemical and molecular studies of microbial mats collected from Elkhorn Slough, Monterey Bay, California, we characterized the mechanisms of H(2) production and identified a dominant hydrogenogen. Net production of H(2) was observed within the upper photosynthetic layer (0-2 mm) of the mats under dark and anoxic conditions. Pyrosequencing of rRNA gene libraries generated from this layer demonstrated the presence of 64 phyla, with Bacteriodetes, Cyanobacteria and Proteobacteria dominating the sequences. Sequencing of rRNA transcripts obtained from this layer demonstrated that Cyanobacteria dominated rRNA transcript pyrotag libraries. An OTU affiliated to Microcoleus spp. was the most abundant OTU in both rRNA gene and transcript libraries. Depriving mats of sunlight resulted in an order of magnitude decrease in subsequent nighttime H(2) production, suggesting that newly fixed carbon is critical to H(2) production. Suppression of nitrogen (N(2))-fixation in the mats did not suppress H(2) production, which indicates that co-metabolic production of H(2) during N(2)-fixation is not an important contributor to H(2) production. Concomitant production of organic acids is consistent with fermentation of recently produced photosynthate as the dominant mode of H(2) production. Analysis of rRNA % transcript:% gene ratios and H(2)-evolving bidirectional [NiFe] hydrogenase % transcript:% gene ratios indicated that Microcoelus spp. are dominant hydrogenogens in the Elkhorn Slough mats.

Abstract

We developed a broad-ranging method for identifying key hydrogen-producing and consuming microorganisms through analysis of hydrogenase gene content and expression in complex anaerobic microbial communities. The method is based on a tiling hydrogenase gene oligonucleotide DNA microarray (Hydrogenase Chip), which implements a high number of probes per gene by tiling probe sequences across genes of interest at 1.67 × -2 × coverage. This design favors the avoidance of false positive gene identification in samples of DNA or RNA extracted from complex microbial communities. We applied this technique to interrogate interspecies hydrogen transfer in complex communities in (i) lab-scale reductive dehalogenating microcosms enabling us to delineate key H(2)-consuming microorganisms, and (ii) hydrogen-generating microbial mats where we found evidence for significant H(2) production by cyanobacteria. Independent quantitative PCR analysis on selected hydrogenase genes showed that this Hydrogenase Chip technique is semiquantitative. We also determined that as microbial community complexity increases, specificity must be traded for sensitivity in analyzing data from tiling DNA microarrays.

Abstract

Stability and resistance to dissolution are key features of microbial biofilms. How these macroscopic properties are determined by the physiological state of individual biofilm cells in their local physical-chemical and cellular environment is largely unknown. In order to obtain molecular and energetic insight into biofilm stability, we investigated whether maintenance of biofilm stability is an energy-dependent process and whether transcription and/or translation is required for biofilm dissolution. We found that in 12-hour-old Shewanella oneidensis MR-1 biofilms, a reduction in cellular ATP concentration, induced either by oxygen deprivation or by addition of the inhibitor of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP), dinitrophenol (DNP), or CN(-), resulted in massive dissolution. In 60-hour-old biofilms, the extent of uncoupler-induced cell loss was strongly attenuated, indicating that the integrity of older biofilms is maintained by means other than those operating in younger biofilms. In experiments with 12-hour-old biofilms, the transcriptional and translational inhibitors rifampin, tetracycline, and erythromycin were found to be ineffective in preventing energy starvation-induced detachment, suggesting that neither transcription nor translation is required for this process. Biofilms of Vibrio cholerae were also induced to dissolve upon CCCP addition to an extent similar to that in S. oneidensis. However, Pseudomonas aeruginosa and P. putida biofilms remained insensitive to CCCP addition. Collectively, our data show that metabolic energy is directly or indirectly required for maintaining cell attachment, and this may represent a common but not ubiquitous mechanism for stability of microbial biofilms.

Abstract

Vinyl chloride is a widespread groundwater pollutant and Group 1 carcinogen. A previous comparative genomic analysis revealed that the vinyl chloride reductase operon, vcrABC, of Dehalococcoides sp. strain VS is embedded in a horizontally-acquired genomic island that integrated at the single-copy tmRNA gene, ssrA.We targeted conserved positions in available genomic islands to amplify and sequence four additional vcrABC -containing genomic islands from previously-unsequenced vinyl chloride respiring Dehalococcoides enrichments. We identified a total of 31 ssrA-specific genomic islands from Dehalococcoides genomic data, accounting for 47 reductive dehalogenase homologous genes and many other non-core genes. Sixteen of these genomic islands contain a syntenic module of integration-associated genes located adjacent to the predicted site of integration, and among these islands, eight contain vcrABC as genetic 'cargo'. These eight vcrABC -containing genomic islands are syntenic across their ~12 kbp length, but have two phylogenetically discordant segments that unambiguously differentiate the integration module from the vcrABC cargo. Using available Dehalococcoides phylogenomic data we estimate that these ssrA-specific genomic islands are at least as old as the Dehalococcoides group itself, which in turn is much older than human civilization.The vcrABC -containing genomic islands are a recently-acquired subset of a diverse collection of ssrA-specific mobile elements that are a major contributor to strain-level diversity in Dehalococcoides, and may have been throughout its evolution. The high similarity between vcrABC sequences is quantitatively consistent with recent horizontal acquisition driven by ~100 years of industrial pollution with chlorinated ethenes.

Abstract

The resistance of biofilms to conventional antibiotics complicates the treatment of chronic cystic fibrosis (CF). We investigated the effects of peptoids, peptides, and conventional antibiotics on the biomass and cell viability within Pseudomonas aeruginosa biofilms. At their MICs, peptoids 1 and 1-C13(4mer) caused maximum reductions in biomass and cell viability, respectively. These results suggest that peptoids of this class could be worth exploring for the treatment of pulmonary infections occurring in CF patients.

Abstract

The gammaproteobacterium Shewanella oneidensis MR-1 utilizes a complex electron transfer network composed primarily of c-type cytochromes to respire under anoxic conditions a variety of compounds, including fumarate, nitrate, and dimethyl sulfoxide (DMSO), in addition to the minerals Fe(III) and Mn(IV). Central to several respiratory pathways is CymA, a cytoplasmic membrane-bound tetraheme c-type cytochrome that functions as the major hydroquinone dehydrogenase. To investigate functional redundancy and plasticity in S. oneidensis MR-1 electron transport, we isolated ?cymA suppressor mutants and characterized one biochemically and genetically. Interestingly, in the characterized ?cymA suppressor mutant, respiration of fumarate, ferric citrate, and DMSO was restored but that of nitrate was not. The suppression was found to be due to transcriptional activation of sirC and sirD, encoding a periplasmic iron sulfur protein and an integral membrane hydroquinone dehydrogenase, respectively. Biochemical in vitro reconstitution experiments confirmed electron transport between formate and fumarate via fumarate reductase by suppressor membrane fractions. The suppression was found to be caused by insertion of an ISSod1 element upstream of the sirCD transcriptional start site, generating a novel, constitutively active hybrid promoter. This work revealed that adaptation of an alternative electron transfer pathway from quinol to terminal oxidoreductases independent of CymA occurs rapidly in S. oneidensis MR-1.

Abstract

The GGDEF domain protein MxdA, which is important for biofilm formation in Shewanella oneidensis MR-1, was hypothesized to possess diguanylate cyclase activity. Here, we demonstrate that while MxdA controls the cellular level of c-di-GMP in S. oneidensis, it modulates the c-di-GMP pool indirectly.

Abstract

Irritable bowel syndrome (IBS) is a chronic, episodic gastrointestinal disorder that is prevalent in a significant fraction of western human populations; and changes in the microbiota of the large bowel have been implicated in the pathology of the disease.Using a novel comprehensive, high-density DNA microarray (PhyloChip) we performed a phylogenetic analysis of the microbial community of the large bowel in a rat model in which intracolonic acetic acid in neonates was used to induce long lasting colonic hypersensitivity and decreased stool water content and frequency, representing the equivalent of human constipation-predominant IBS.Our results revealed a significantly increased compositional difference in the microbial communities in rats with neonatal irritation as compared with controls. Even more striking was the dramatic change in the ratio of Firmicutes relative to Bacteroidetes, where neonatally irritated rats were enriched more with Bacteroidetes and also contained a different composition of species within this phylum. Our study also revealed differences at the level of bacterial families and species.The PhyloChip is a useful and convenient method to study enteric microflora. Further, this rat model system may be a useful experimental platform to study the causes and consequences of changes in microbial community composition associated with IBS.

Abstract

This article explains the statistical and computational methodology used to analyze species abundances collected using the LNBL Phylochip in a study of Irritable Bowel Syndrome (IBS) in rats. Some tools already available for the analysis of ordinary microarray data are useful in this type of statistical analysis. For instance in correcting for multiple testing we use Family Wise Error rate control and step-down tests (available in the multtest package). Once the most significant species are chosen we use the hypergeometric tests familiar for testing GO categories to test specific phyla and families. We provide examples of normalization, multivariate projections, batch effect detection and integration of phylogenetic covariation, as well as tree equalization and robustification methods.

Abstract

Type IV pili and a putative EPS biosynthetic gene cluster (mxdABCD) have been implicated previously in biofilm formation in Shewanella oneidensis MR-1. Here, we report that the mannose-sensitive hemagglutinin (MSHA) pilus mediates a reversible, d-mannose-sensitive association of cells to the substratum surface or to other cells that is critical within the first 5 microm of the biofilm from the substratum. The presence of the MSHA pilus alone is insufficient to confer biofilm-forming capacity; its activity, as mediated by the putative pilus retraction motor protein, PilT, is also required. Deletion of pilD, encoding the type IV pili prepilin peptidase, revealed that additional PilD substrate(s) may be involved in biofilm formation beyond the major structural pilin of the MSHA pilus. We also present data showing that the MSHA pilus and mxd genes encode for a complementary set of molecular machineries that constitute the dominant mechanisms enabling biofilm formation in this microorganism under hydrodynamic conditions.

Abstract

A continuous-flow column study was conducted to analyze the reductive dehalogenation of trichloroethene (TCE) with aquifer material with high content of iron oxides. The column was bioaugmented with the Point Mugu (PM) culture, which is a mixed microbial enrichment culture capable of completely transforming TCE to ethene (ETH). We determined whether lactate, formate, or propionate fermentation resulted in more effective dehalogenation. Reductive dehalogenation, fermentation, and sulfate, Fe(III), and Mn(IV) reduction were all exhibited within the column. Different steady-states of dehalogenation were achieved based on the concentration of substrates added, with effective transformation to ETH obtained when ample electron donor equivalents were provided. Most of the metabolic reducing equivalents were channeled to sulfate, Fe(III), and Mn(IV) reduction. When similar electron reducing equivalents were added, the most effective dehalogenation was achieved with formate, with 14% of the electron equivalents going towards dehalogenation reactions, compared to 6.5% for lactate and 9.6% for propionate. Effective dehalogenation was maintained over 1000 days of column operation. Over 90% of electron equivalents added could be accounted for by the different electron accepting processes in the column, with 50% associated with soluble and precipitated Fe(II) and Mn(II). Bulk Fe(III) and Mn(IV) reduction was rather associated with lactate and propionate addition than formate addition. Sulfate reduction was a competing electron acceptor reaction with all three electron donors. DNA was extracted from solid coupon samples obtained during the course of the experiment and analyzed using 16S rRNA gene clone libraries and quantitative PCR. Lactate and propionate addition resulted in a significant increase in Geobacter, Spirochaetes, and Desulfitobacterium phylotypes relative to "Dehalococcoides" when compared to formate addition. Results from the molecular biological analyses support chemical observations that a greater percentage of the electron donor addition was channeled to Fe(III) reduction when lactate and propionate were added compared to formate, and formate was more effective than lactate in supporting dehalogenation. The results demonstrate the importance of electron donor selection and competing electron acceptor reactions when implementing reductive dehalogenation remediation technologies.

Abstract

Francisella tularensis, the zoonotic cause of tularemia, can infect numerous mammals and other eukaryotes. Although studying F. tularensis pathogenesis is essential to comprehending disease, mammalian infection is just one step in the ecology of Francisella species. F. tularensis has been isolated from aquatic environments and arthropod vectors, environments in which chitin could serve as a potential carbon source and as a surface for attachment and growth. We show that F. tularensis subsp. novicida forms biofilms during the colonization of chitin surfaces. The ability of F. tularensis to persist using chitin as a sole carbon source is dependent on chitinases, since mutants lacking chiA or chiB are attenuated for chitin colonization and biofilm formation in the absence of exogenous sugar. A genetic screen for biofilm mutants identified the Sec translocon export pathway and 14 secreted proteins. We show that these genes are important for initial attachment during biofilm formation. We generated defined deletion mutants by targeting two chaperone genes (secB1 and secB2) involved in Sec-dependent secretion and four genes that encode putative secreted proteins. All of the mutants were deficient in attachment to polystyrene and chitin surfaces and for biofilm formation compared to wild-type F. novicida. In contrast, mutations in the Sec translocon and secreted factors did not affect virulence. Our data suggest that biofilm formation by F. tularensis promotes persistence on chitin surfaces. Further study of the interaction of F. tularensis with the chitin microenvironment may provide insight into the environmental survival and transmission mechanisms of this pathogen.

Abstract

Dissimilatory microbial reduction of insoluble Fe(III) oxides is a geochemically and ecologically important process which involves the transfer of cellular, respiratory electrons from the cytoplasmic membrane to insoluble, extracellular, mineral-phase electron acceptors. In this paper evidence is provided for the function of the periplasmic fumarate reductase FccA and the decaheme c-type cytochrome MtrA in periplasmic electron transfer reactions in the gammaproteobacterium Shewanella oneidensis. Both proteins are abundant in the periplasm of ferric citrate-reducing S. oneidensis cells. In vitro fumarate reductase FccA and c-type cytochrome MtrA were reduced by the cytoplasmic membrane-bound protein CymA. Electron transfer between CymA and MtrA was 1.4-fold faster than the CymA-catalyzed reduction of FccA. Further experiments showing a bidirectional electron transfer between FccA and MtrA provided evidence for an electron transfer network in the periplasmic space of S. oneidensis. Hence, FccA could function in both the electron transport to fumarate and via MtrA to mineral-phase Fe(III). Growth experiments with a DeltafccA deletion mutant suggest a role of FccA as a transient electron storage protein.

Abstract

Vinyl chloride (VC) is a human carcinogen and widespread priority pollutant. Here we report the first, to our knowledge, complete genome sequences of microorganisms able to respire VC, Dehalococcoides sp. strains VS and BAV1. Notably, the respective VC reductase encoding genes, vcrAB and bvcAB, were found embedded in distinct genomic islands (GEIs) with different predicted integration sites, suggesting that these genes were acquired horizontally and independently by distinct mechanisms. A comparative analysis that included two previously sequenced Dehalococcoides genomes revealed a contextually conserved core that is interrupted by two high plasticity regions (HPRs) near the Ori. These HPRs contain the majority of GEIs and strain-specific genes identified in the four Dehalococcoides genomes, an elevated number of repeated elements including insertion sequences (IS), as well as 91 of 96 rdhAB, genes that putatively encode terminal reductases in organohalide respiration. Only three core rdhA orthologous groups were identified, and only one of these groups is supported by synteny. The low number of core rdhAB, contrasted with the high rdhAB numbers per genome (up to 36 in strain VS), as well as their colocalization with GEIs and other signatures for horizontal transfer, suggests that niche adaptation via organohalide respiration is a fundamental ecological strategy in Dehalococccoides. This adaptation has been exacted through multiple mechanisms of recombination that are mainly confined within HPRs of an otherwise remarkably stable, syntenic, streamlined genome among the smallest of any free-living microorganism.

Abstract

We investigated the distribution and activity of chloroethene-degrading microorganisms and associated functional genes during reductive dehalogenation of tetrachloroethene to ethene in a laboratory continuous-flow column. Using real-time PCR, we quantified "Dehalococcoides" species 16S rRNA and chloroethene-reductive dehalogenase (RDase) genes (pceA, tceA, vcrA, and bvcA) in nucleic acid extracts from different sections of the column. Dehalococcoides 16S rRNA gene copies were highest at the inflow port [(3.6 +/- 0.6) x 10(6) (mean +/- standard deviation) per gram soil] where the electron donor and acceptor were introduced into the column. The highest transcript numbers for tceA, vcrA, and bvcA were detected 5 to 10 cm from the column inflow. bvcA was the most highly expressed of all RDase genes and the only vinyl chloride reductase-encoding transcript detectable close to the column outflow. Interestingly, no expression of pceA was detected in the column, despite the presence of the genes in the microbial community throughout the column. By comparing the 16S rRNA gene copy numbers to the sum of all four RDase genes, we found that 50% of the Dehalococcoides population in the first part of the column did not contain either one of the known chloroethene RDase genes. Analysis of 16S rRNA gene clone libraries from both ends of the flow column revealed a microbial community dominated by members of Firmicutes and Actinobacteria. Higher clone sequence diversity was observed near the column outflow. The results presented have implications for our understanding of the ecophysiology of reductively dehalogenating Dehalococcoides spp. and their role in bioremediation of chloroethenes.

Abstract

A continuous-flow anaerobic column experiment was conducted to evaluate the reductive dechlorination of tetrachloroethene (PCE) in Hanford aquifer material after bioaugmentation with the Evanite (EV) culture. An influent PCE concentration of 0.09 mM was transformed to vinyl chloride (VC) and ethene (ETH) within a hydraulic residence time of 1.3 days. The experimental breakthrough curves were described by the one-dimensional two-site-nonequilibrium transport model. PCE dechlorination was observed after bioaugmentation and after the lactate concentration was increased from 0.35 to 0.67 mM. At the onset of reductive dehalogenation, cis-dichloroethene (c-DCE) concentrations in the column effluent exceeded the influent PCE concentration indicating enhanced PCE desorption and transformation. When the lactate concentration was increased to 1.34 mM, c-DCE reduction to vinyl chloride (VC) and ethene (ETH) occurred. Spatial rates of PCE and VC transformation were determined in batch-incubated microcosms constructed with aquifer samples obtained from the column. PCE transformation rates were highest in the first 5 cm from the column inlet and decreased towards the column effluent. Dehalococcoides cell numbers dropped from approximately 73.5% of the total Bacterial population in the original inocula, to about 0.5% to 4% throughout the column. The results were consistent with estimates of electron donor utilization, with 4% going towards dehalogenation reactions.

Abstract

Bacteria of the genus Shewanella are known for their versatile electron-accepting capacities, which allow them to couple the decomposition of organic matter to the reduction of the various terminal electron acceptors that they encounter in their stratified environments. Owing to their diverse metabolic capabilities, shewanellae are important for carbon cycling and have considerable potential for the remediation of contaminated environments and use in microbial fuel cells. Systems-level analysis of the model species Shewanella oneidensis MR-1 and other members of this genus has provided new insights into the signal-transduction proteins, regulators, and metabolic and respiratory subsystems that govern the remarkable versatility of the shewanellae.

Abstract

The NASA Astrobiology Roadmap provides guidance for research and technology development across the NASA enterprises that encompass the space, Earth, and biological sciences. The ongoing development of astrobiology roadmaps embodies the contributions of diverse scientists and technologists from government, universities, and private institutions. The Roadmap addresses three basic questions: how does life begin and evolve, does life exist elsewhere in the universe, and what is the future of life on Earth and beyond? Seven Science Goals outline the following key domains of investigation: understanding the nature and distribution of habitable environments in the universe, exploring for habitable environments and life in our own Solar System, understanding the emergence of life, determining how early life on Earth interacted and evolved with its changing environment, understanding the evolutionary mechanisms and environmental limits of life, determining the principles that will shape life in the future, and recognizing signatures of life on other worlds and on early Earth. For each of these goals, Science Objectives outline more specific high priority efforts for the next three to five years. These eighteen objectives are being integrated with NASA strategic planning.

Abstract

Over geological time scales, microbial reduction of chelated Fe(III) or Fe(III) minerals has profoundly affected today's composition of our bio- and geosphere. However, the electron transfer reactions that are specific and defining for dissimilatory iron(III)-reducing (DIR) bacteria are not well understood. Using a synthetic biology approach involving the reconstruction of the putative electron transport chain of the DIR bacterium Shewanella oneidensis MR-1 in Escherichia coli, we showed that expression of cymA was necessary and sufficient to convert E. coli into a DIR bacterium. In intact cells, the Fe(III)-reducing activity was limited to Fe(III) NTA as electron acceptor. In vitro biochemical analysis indicated that CymA, which is a cytoplasmic membrane-associated tetrahaem c-type cytochrome, carries reductase activity towards Fe(III) NTA, Fe(III) citrate, as well as to AQDS, a humic acid analogue. The in vitro specific activities of Fe(III) citrate reductase and AQDS reductase of E. coli spheroplasts were 10x and 30x higher, respectively, relative to the specific rates observed in intact cells, suggesting that access of chelated and insoluble forms of Fe(III) and AQDS is restricted in whole cells. Interestingly, the E. coli CymA orthologue NapC also carried ferric reductase activity. Our data support the argument that the biochemical mechanism of Fe(III) reduction per se was not the key innovation leading to environmental relevant DIR bacteria. Rather, the evolution of an extension of the electron transfer pathway from the Fe(III) reductase CymA to the cell surface via a system of periplasmic and outer membrane cytochrome proteins enabled access to diffusion-impaired electron acceptors.

Abstract

To examine phylogenetic identity and metabolic activity of individual cells in complex microbial communities, we developed a method which combines rRNA-based in situ hybridization with stable isotope imaging based on nanometer-scale secondary-ion mass spectrometry (NanoSIMS). Fluorine or bromine atoms were introduced into cells via 16S rRNA-targeted probes, which enabled phylogenetic identification of individual cells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified the current catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogen-containing fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, we obtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundance of fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold and allowed us to distinguish target from non-target cells in NanoSIMS fluorine or bromine images. The method was optimized on single cells of axenic Escherichia coli and Vibrio cholerae cultures. EL-FISH/NanoSIMS was then applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacterium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggregates obtained from human oral biofilms. In both samples, we found evidence for metabolic interactions by visualizing the fate of substrates labeled with (13)C-carbon and (15)N-nitrogen, while individual cells were identified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studies of the ecophysiology of known and uncultured microorganisms in complex environments and communities.

Abstract

Microbial biofilms are governed by an intricate interplay between physical-chemical factors and the physiological and genetic properties of the inhabiting microbes. Many of the physiological traits that are exhibited in a biofilm environment have been observed and studied previously in detail in planktonic cultures. However, their differential and combinatorial phenotypic expression in distinct subpopulations localized to different regions in a biofilm is the cause for the overall biofilm heterogeneity. In this chapter, the causes and consequences of this interplay are elaborated with a special focus on processes controlling biofilm stability and dispersal.

Abstract

The natural life cycle of Vibrio cholerae involves the transitioning of cells between different environmental surfaces such as the chitinous shell of Crustaceae and the epithelial layer of the human intestine. Previous studies using static biofilm systems showed a strict dependence of biofilm formation on the vps and lux genes, which are essential for exopolysaccharide formation and cell-cell signaling, respectively. The authors' report here that in biofilms grown under hydrodynamic conditions, DeltavpsA and DeltaluxO mutants of V. cholerae do form pronounced, three-dimensional biofilms that resemble all aspects of wild-type biofilms. By genetic experiments, it was shown that in hydrodynamically grown biofilms this independence of vpsA is due to the expression of rpoS, which is a negative regulator of vpsA expression. Biofilms also underwent substantial dissolution after 96 h that could be induced by a simple stop of medium flow. The studies indicate that metabolic conditions control the reversible attachment of cells to the biofilm matrix and are key in regulating biofilm cell physiology via RpoS. Furthermore, the results redefine the roles of vps and quorum-sensing in V. cholerae biofilms.

Abstract

Vinyl chloride reductases (VC-RDase) are the key enzymes for complete microbial reductive dehalogenation of chloroethenes, including the groundwater pollutants tetrachloroethene and trichloroethene. Analysis of the codon usage of the VC-RDase genes vcrA and bvcA showed that these genes are highly unusual and are characterized by a low G+C fraction at the third position. The third position of codons in VC-RDase genes is biased toward the nucleotide T, even though available Dehalococcoides genome sequences indicate the absence of any tRNAs matching codons that end in T. The comparatively high level of abnormality in the codon usage of VC-RDase genes suggests an evolutionary history that is different from that of most other Dehalococcoides genes.

Abstract

Shewanella oneidensis MR-1 is a facultative sediment microorganism which uses diverse compounds, such as oxygen and fumarate, as well as insoluble Fe(III) and Mn(IV) as electron acceptors. The electron donor spectrum is more limited and includes metabolic end products of primary fermenting bacteria, such as lactate, formate, and hydrogen. While the utilization of hydrogen as an electron donor has been described previously, we report here the formation of hydrogen from pyruvate under anaerobic, stationary-phase conditions in the absence of an external electron acceptor. Genes for the two S. oneidensis MR-1 hydrogenases, hydA, encoding a periplasmic [Fe-Fe] hydrogenase, and hyaB, encoding a periplasmic [Ni-Fe] hydrogenase, were found to be expressed only under anaerobic conditions during early exponential growth and into stationary-phase growth. Analyses of DeltahydA, DeltahyaB, and DeltahydA DeltahyaB in-frame-deletion mutants indicated that HydA functions primarily as a hydrogen-forming hydrogenase while HyaB has a bifunctional role and represents the dominant hydrogenase activity under the experimental conditions tested. Based on results from physiological and genetic experiments, we propose that hydrogen is formed from pyruvate by multiple parallel pathways, one pathway involving formate as an intermediate, pyruvate-formate lyase, and formate-hydrogen lyase, comprised of HydA hydrogenase and formate dehydrogenase, and a formate-independent pathway involving pyruvate dehydrogenase. A reverse electron transport chain is potentially involved in a formate-hydrogen lyase-independent pathway. While pyruvate does not support a fermentative mode of growth in this microorganism, pyruvate, in the absence of an electron acceptor, increased cell viability in anaerobic, stationary-phase cultures, suggesting a role in the survival of S. oneidensis MR-1 under stationary-phase conditions.

Abstract

Stability and resilience against environmental perturbations are critical properties of medical and environmental biofilms and pose important targets for their control. Biofilm stability is determined by two mutually exclusive processes: attachment of cells to and detachment from the biofilm matrix. Using Shewanella oneidensis MR-1, an environmentally versatile, Fe(III) and Mn(IV) mineral-reducing microorganism, we identified mxdABCD as a new set of genes essential for formation of a three-dimensional biofilm. Molecular analysis revealed that mxdA encodes a cyclic bis(3',5')guanylic acid (cyclic di-GMP)-forming enzyme with an unusual GGDEF motif, i.e., NVDEF, which is essential for its function. mxdB encodes a putative membrane-associated glycosyl transferase. Both genes are essential for matrix attachment. The attachment-deficient phenotype of a DeltamxdA mutant was rescued by ectopic expression of VCA0956, encoding another diguanylate cyclase. Interestingly, a rapid cellular detachment from the biofilm occurred upon induction of yhjH, a gene encoding an enzyme that has been shown to have phosphodiesterase activity. In this way, it was possible to bypass the previously identified sudden depletion of molecular oxygen as an environmental trigger to induce biofilm dissolution. We propose a model for c-di-GMP as a key intracellular regulator for controlling biofilm stability by shifting the state of a biofilm cell between attachment and detachment in a concentration-dependent manner.

Abstract

Predicted highly expressed (PHX) genes are compared for 16 gamma-proteobacteria and their similarities and differences are interpreted with respect to known or predicted physiological characteristics of the organisms. Predicted highly expressed genes often reflect the organism's predominant lifestyle, habitat, nutrition sources and metabolic propensities. This technique allows to predict principal metabolic activities of the microorganisms operating in their natural habitats. Among our findings is an unusually high number of PHX enzymes acting in cell wall biosynthesis, amino acid biosynthesis and replication in the ant endosymbiont Blochmannia floridanus. We ascribe the abundance of these PHX genes to specific aspects of the relationship between the bacterium and its host. Xanthomonas campestris is unique with a very high number of PHX genes acting in flagellum biosynthesis, which may play a special role during its pathogenicity. Shewanella oneidensis possesses three protein complexes which all can function as complex I in the respiratory chain but only the Na(+)-transporting NADH:ubiquinone oxidoreductase nqr-2 operon is PHX. The PHX genes of Vibrio parahaemolyticus are consistent with the microorganism's adaptation to extremely fast growth rates. Comparative analysis of PHX genes from complex environmental genomic sequences as well as from uncultured pathogenic microbes can provide a novel, useful tool to predict global flux of matter and key intermediates.

Abstract

Vibrio cholerae, the etiological agent of the diarrheal illness cholera, can kill an infected adult in 24 h. V.cholerae lives as an autochthonous microbe in estuaries, rivers and coastal waters. A better understanding of its metabolic pathways will assist the development of more effective treatments and will provide a deeper understanding of how this bacterium persists in natural aquatic habitats. Using the completed V.cholerae genome sequence and PathoLogic software, we created VchoCyc, a pathway-genome database that predicted 171 likely metabolic pathways in the bacterium. We report here experimental evidence supporting the computationally predicted pathways. The evidence comes from microarray gene expression studies of V.cholerae in the stools of three cholera patients [D. S. Merrell, S. M. Butler, F. Qadri, N. A. Dolganov, A. Alam, M. B. Cohen, S. B. Calderwood, G. K. Schoolnik and A. Camilli (2002) Nature, 417, 642-645.], from gene expression studies in minimal growth conditions and LB rich medium, and from clinical tests that identify V.cholerae. Expression data provide evidence supporting 92 (53%) of the 171 pathways. The clinical tests provide evidence supporting seven pathways, with six pathways supported by both methods. VchoCyc provides biologists with a useful tool for analyzing this organism's metabolic and genomic information, which could lead to potential insights into new anti-bacterial agents. VchoCyc is available in the BioCyc database collection (http://BioCyc.org).

Abstract

Active detachment of cells from microbial biofilms is a critical yet poorly understood step in biofilm development. We discovered that detachment of cells from biofilms of Shewanella oneidensis MR-1 can be induced by arresting the medium flow in a hydrodynamic biofilm system. Induction of detachment was rapid, and substantial biofilm dispersal started as soon as 5 min after the stop of flow. We developed a confocal laser scanning microscopy-based assay to quantify detachment. The extent of biomass loss was found to be dependent on the time interval of flow stop and on the thickness of the biofilm. Up to 80% of the biomass of 16-h-old biofilms could be induced to detach. High-resolution microscopy studies revealed that detachment was associated with an overall loosening of the biofilm structure and a release of individual cells or small cell clusters. Swimming motility was not required for detachment. Although the loosening of cells from the biofilm structure was observed evenly throughout thin biofilms, the most pronounced detachment in thicker biofilms occurred in regions exposed to the flow of medium, suggesting a metabolic control of detachability. Deconvolution of the factors associated with the stop of medium flow revealed that a sudden decrease in oxygen tension is the predominant trigger for initiating detachment of individual cells. In contrast, carbon limitation did not trigger any substantial detachment, suggesting a physiological link between oxygen sensing or metabolism and detachment. In-frame deletions were introduced into genes encoding the known and putative global transcriptional regulators ArcA, CRP, and EtrA (FNR), which respond to changes in oxygen tension in S. oneidensis MR-1. Biofilms of null mutants in arcA and crp were severely impacted in the stop-of-flow-induced detachment response, suggesting a role for these genes in regulation of detachment. In contrast, an DeltaetrA mutant displayed a variable detachment phenotype. From this genetic evidence we conclude that detachment is a biologically controlled process and that a rapid change in oxygen concentration is a critical factor in detachment and, consequently, in dispersal of S. oneidensis cells from biofilms. Similar mechanisms might also operate in other bacteria.

Dynamics and control of biofilms of the oligotrophic bacterium Caulobacter crescentusJOURNAL OF BACTERIOLOGYEntcheva-Dimitrov, P., Spormann, A. M.2004; 186 (24): 8254-8266

Abstract

Caulobacter crescentus is an oligotrophic alpha-proteobacterium with a complex cell cycle involving sessile-stalked and piliated, flagellated swarmer cells. Because the natural lifestyle of C. crescentus intrinsically involves a surface-associated, sessile state, we investigated the dynamics and control of C. crescentus biofilms developing on glass surfaces in a hydrodynamic system. In contrast to biofilms of the well-studied Pseudomonas aeruginosa, Escherichia coli, and Vibrio cholerae, C. crescentus CB15 cells form biphasic biofilms, consisting predominantly of a cell monolayer biofilm and a biofilm containing densely packed, mushroom-shaped structures. Based on comparisons between the C. crescentus strain CB15 wild type and its holdfast (hfsA; DeltaCC0095), pili (DeltapilA-cpaF::Omegaaac3), motility (motA), flagellum (flgH) mutants, and a double mutant lacking holdfast and flagellum (hfsA; flgH), a model for biofilm formation in C. crescentus is proposed. For both biofilm forms, the holdfast structure at the tip of a stalked cell is crucial for mediating the initial attachment. Swimming motility by means of the single polar flagellum enhances initial attachment and enables progeny swarmer cells to escape from the monolayer biofilm. The flagellum structure also contributes to maintaining the mushroom structure. Type IV pili enhance but are not absolutely required for the initial adhesion phase. However, pili are essential for forming and maintaining the well-defined three-dimensional mushroom-shaped biofilm. The involvement of pili in mushroom architecture is a novel function for type IV pili in C. crescentus. These unique biofilm features demonstrate a spatial diversification of the C. crescentus population into a sessile, "stem cell"-like subpopulation (monolayer biofilm), which generates progeny cells capable of exploring the aqueous, oligotrophic environment by swimming motility and a subpopulation accumulating in large mushroom structures.

Abstract

Shewanella oneidensis MR-1 is a facultative Fe(III)- and Mn(IV)-reducing microorganism and serves as a model for studying microbially induced dissolution of Fe or Mn oxide minerals as well as biogeochemical cycles. In soil and sediment environments, S. oneidensis biofilms form on mineral surfaces and are critical for mediating the metabolic interaction between this microbe and insoluble metal oxide phases. In order to develop an understanding of the molecular basis of biofilm formation, we investigated S. oneidensis biofilms developing on glass surfaces in a hydrodynamic flow chamber system. After initial attachment, growth of microcolonies and lateral spreading of biofilm cells on the surface occurred simultaneously within the first 24 h. Once surface coverage was almost complete, biofilm development proceeded with extensive vertical growth, resulting in formation of towering structures giving rise to pronounced three-dimensional architecture. Biofilm development was found to be dependent on the nutrient conditions, suggesting a metabolic control. In global transposon mutagenesis, 173 insertion mutants out of 15,000 mutants screened were identified carrying defects in initial attachment and/or early stages in biofilm formation. Seventy-one of those mutants exhibited a nonswimming phenotype, suggesting a role of swimming motility or motility elements in biofilm formation. Disruption mutations in motility genes (flhB, fliK, and pomA), however, did not affect initial attachment but affected progression of biofilm development into pronounced three-dimensional architecture. In contrast, mutants defective in mannose-sensitive hemagglutinin type IV pilus biosynthesis and in pilus retraction (pilT) showed severe defects in adhesion to abiotic surfaces and biofilm formation, respectively. The results provide a basis for understanding microbe-mineral interactions in natural environments.

Abstract

Reductive dehalogenation of tetrachloroethene (PCE), trichloroethene (TCE), cis-1,2-dichloroethene (DCE), and vinyl chloride (VC) was examined in four cultures containing Dehalococcoides-like microorganisms. Dechlorination and growth kinetics were compared using a Monod growth-rate model for multiple electron acceptor usage with competition. Included were the Victoria mixed culture containing Dehalococcoides species strain VS (from Victoria, TX), the mixed culture KB-1/VC (from southern Ontario), the Pinellas mixed culture (from Pinellas, FL), and D. ethenogenes strain 195. All cultures, with the exception of D. ethenogenes strain 195, grew with VC as catabolic electron acceptor. A dilution method was developed that allows a valid comparison to be made of dehalogenating kinetics between different mixed cultures. Using this procedure, maximum growth rates on VC were found to be similar for strain VS and KB-1/VC (0.42-0.49 +/- 0.02 d(-1)) but slower for the Pinellas culture (0.28 +/- 0.01 d(-1)). The 16S rRNA gene sequences were determined to ensure that no cross contamination between cultures had occurred. Following enrichment of the VC dechlorinating microorganisms on VC, the cultures were amended with DCE, TCE, or PCE. The three mixed cultures failed to dechlorinate PCE or did so very slowly. However, the dilution technique indicated that all experienced growth on TCE and DCE as well as on VC. Maximum growth rates on DCE alone were quite similar (0.43-0.46 d(-1)), while the Pinellas culture grew faster on TCE alone (0.49 d(-1)) than did the other two mixed cultures (0.33-0.35 d(-1)). Half-velocity and inhibition constants for growth on TCE were also determined for the three mixed cultures; both constants were found to be essentially equal and the same for the different cultures, varying between only 8.6 and 10.5 microM. The ability of the strain VS, KB-1/VC, and Pinellas cultures to utilize TCE rapidly with conversion to ethene is quite different from that of any other reported microorganism. It was separately confirmed with more traditional cell-counting techniques that strain VS coupled TCE, as well as DCE and VC, utilization with growth. This is the first report of an organism obtaining energy for growth through every step in the reduction of TCE to ethene. Also, as suggested by the dilution technique, the dehalogenating organisms in the KB-1/VC and Pinellas cultures appear to obtain growth from TCE utilization as well. Such ability to grow while dehalogenating TCE to ethene will be an important advantage for their use in bioaugmentation.

Abstract

Reductive dehalogenation of vinyl chloride (VC) to ethene is the key step in complete anaerobic degradation of chlorinated ethenes. VC-reductive dehalogenase was partially purified from a highly enriched culture of the VC-respiring Dehalococcoides sp. strain VS. The enzyme reduced VC and all dichloroethene (DCE) isomers, but not tetrachloroethene (PCE) or trichloroethene (TCE), at high rates. By using reversed genetics, the corresponding gene (vcrA) was isolated and characterized. Based on the predicted amino acid sequence, VC reductase is a novel member of the family of corrinoid/iron-sulfur cluster containing reductive dehalogenases. The vcrA gene was found to be cotranscribed with vcrB, encoding a small hydrophobic protein presumably acting as membrane anchor for VC reductase, and vcrC, encoding a protein with similarity to transcriptional regulators of the NosR/NirI family. The vcrAB genes were subsequently found to be present and expressed in other cultures containing VC-respiring Dehalococcoides organisms and could be detected in water samples from a field site contaminated with chlorinated ethenes. Therefore, the vcrA gene identified here may be a useful molecular target for evaluating, predicting, and monitoring in situ reductive VC dehalogenation.

Abstract

Transformation of carbon tetrachloride (CT) by Shewanella oneidensis MR-1 has been proposed to involve the anaerobic respiratory-chain component menaquinone. To investigate this hypothesis a series of menaquinone mutants were constructed. The menF mutant is blocked at the start of the menaquinone biosynthetic pathway. The menB, menA and menG mutants are all blocked towards the end of the pathway, being unable to produce 1,4-dihydroxy-2-naphthoic acid (DHNA), demethyl-menaquinone and menaquinone, respectively. Aerobically grown mutants unable to produce the menaquinone precursor DHNA (menF and menB mutants) showed a distinctly different CT transformation profile than mutants able to produce DHNA but unable to produce menaquinone (menA and menG mutants). While DHNA did not reduce CT in an abiotic assay, the addition of DHNA to the menF and menB mutants restored normal CT transformation activity. We conclude that a derivative of DHNA, that is distinct from menaquinone, is involved in the reduction of CT by aerobically grown S. oneidensis MR-1. When cells were grown anaerobically with trimethylamine-N-oxide as the terminal electron acceptor, all the menaquinone mutants showed wild-type levels of CT reduction. We conclude that S. oneidensis MR-1 produces two different factors capable of dehalogenating CT. The factor produced under anaerobic growth conditions is not a product of the menaquinone biosynthetic pathway.

Abstract

The reductive dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE) at contaminated sites often results in the accumulation of cis-1,2-dichloroethene (DCE) and vinyl chloride (VC), rather than the nonhazardous end product ethene. This accumulation may be caused by the absence of appropriate microorganisms, insufficient supply of donor substrate, or reaction kinetic limitations. Here, we address the issue of reaction kinetic limitations by investigating the effect of limiting substrate concentrations (electron donor and acceptor) on DCE and VC dechlorination kinetics and microorganism growth by bacterium VS. For this, a model based on Monod kinetics, but also accounting for competition between electron acceptors and the effect of low electron donor and acceptor concentrations (dual-substrate kinetics), was examined. Competitive coefficients for VC (7.8 +/- 1.5 microM) and DCE (3.6 +/- 1.1 microM) were obtained and included in the model. The half velocity coefficient for hydrogen, the electron donor, was experimentally determined (7 +/- 2 nM) through investigating dechlorination over different substrate concentrations. This complete model was then used, along with experimental data, to determine substrate concentrations at which the dechlorinating microorganisms would be in net decay. Notably, the model indicates net decay will result if the total electron acceptor concentration (DCE plus VC) is below 0.7 microM, regardless of electron donor levels. The ability to achieve sustainable bioremediation to acceptable levels can be greatly influenced by this threshold level.

Abstract

The NASA Astrobiology Roadmap provides guidance for research and technology development across the NASA enterprises that encompass the space, Earth, and biological sciences. The ongoing development of astrobiology roadmaps embodies the contributions of diverse scientists and technologists from government, universities, and private institutions. The Roadmap addresses three basic questions: How does life begin and evolve, does life exist elsewhere in the universe, and what is the future of life on Earth and beyond? Seven Science Goals outline the following key domains of investigation: understanding the nature and distribution of habitable environments in the universe, exploring for habitable environments and life in our own solar system, understanding the emergence of life, determining how early life on Earth interacted and evolved with its changing environment, understanding the evolutionary mechanisms and environmental limits of life, determining the principles that will shape life in the future, and recognizing signatures of life on other worlds and on early Earth. For each of these goals, Science Objectives outline more specific high-priority efforts for the next 3-5 years. These 18 objectives are being integrated with NASA strategic planning.

Abstract

Bacterial-mineral composites are important in the retention of heavy metals such as Pb due to their large sorption capacity under a wide range of environmental conditions. However, the partitioning of heavy metals between components in such composites is not probed directly. Using Burkholderia cepacia biofilms coated with goethite (alpha-FeOOH) particles, the partitioning of Pb(II) between the biological and iron-(oxyhydr)oxide surfaces has been measured using an X-ray spectroscopic approach. EXAFS spectra were fit to quantitatively determine the fraction of Pb(II) associated with each component as a function of pH and [Pb]. At pH < 5.5, at least 50% of the total sorbed Pb(II) is associated with the biofilm component, whereas the total uptake within the composite is dominated by goethite (> 70% Pb/goethite) above pH 6. Direct comparison can be made between the amount of Pb(II) bound to each component in the composite vs separate binary systems (i.e., Pb/biofilm or Pb/goethite). At high pH, Pb(II) uptake on the biofilm is dramatically decreased due to competition with the goethite surface. In contrast, Pb uptake on goethite is significantly enhanced at low pH (2-fold increase at pH 5) compared to systems with no complexing ligands. The mode of Pb(II)-binding to the goethite component changes from low to high [Pb]. Structural fitting of the EXAFS spectra collected from 10(-5.6) to 10(-3.6) M [Pb]eq at pH 6 shows that the Pb-goethite surface complexes at low [Pb] are dominated by inner-sphere bidentate, binuclear complexes bridging two adjacent singly coordinated surface oxygens, giving rise to Pb-Fe distances of approximately 3.9 A. At high [Pb], the dominant Pb(II) inner-sphere complexes on the goethite surface shift to bidentate edge-sharing complexes with Pb-Fe distances of approximately 3.3 A.

Abstract

A competitive PCR (cPCR) assay targeting 16S ribosomal DNA was developed to enumerate growth of a Dehalococcoides-like microorganism, bacterium VS, from a mixed culture catalyzing the reductive dehalogenation of cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC), with hydrogen being used as an electron donor. The growth of bacterium VS was found to be coupled to the dehalogenation of VC and cDCE, suggesting unique metabolic capabilities. The average growth yield was (5.2 +/- 1.5) x 10(8) copies of the 16S rRNA gene/ micromol of Cl(-) (number of samples, 10), with VC being used as the electron acceptor and hydrogen as the electron donor. The maximum VC utilization rate (q) was determined to be 7.8 x 10(-10) micromol of Cl(-) (copy(-1) day(-1)), indicating a maximum growth rate of 0.4 day(-1). These average growth yield and q values agree well with values found previously for dechlorinating cultures. Decay coefficients were determined with growth (0.05 day(-1)) and no-growth (0.09 day(-1)) conditions. An important limitation of this cPCR assay was its inability to discriminate between active and inactive cells. This is an essential consideration for kinetic studies.

Abstract

X-ray spectroscopy measurements have been combined with macroscopic uptake data and transmission electron microscopy (TEM) results to show that Pb(II) uptake by Burkholderia cepacia is due to simultaneous sorption and biomineralization processes. X-ray microprobe mapping of B. cepacia biofilms formed on alpha-Al2O3 surfaces shows that Pb(II) is distributed heterogeneously throughout the biofilms because of the formation of Pb "hot spots". EXAFS data and TEM observations show that the enhanced Pb accumulation is due to the formation of nanoscale crystals of pyromorphite (Pb5(PO4)3(OH)) adjacent to the outer-membrane of a fraction of the total population of B. cepacia cells. In contrast, B. cepacia cell suspensions or biofilms that were heat-killed or pretreated with X-rays do not form pyromorphite, which suggests that metabolic activity is required. Precipitation of pyromorphite occurs over several orders of magnitude in [H-] and [Pb] and accounts for approximately 90% of the total Pb uptake below pH 4.5 but only 45-60% at near-neutral pH because of the formation of additional Pb(II) adsorption complexes. Structural fits of Pb L(III) EXAFS data collected for heat-treated cells at near-neutral pH suggest that Pb(II) forms inner-sphere adsorption complexes with carboxyl functional groups in the biofilms.

Abstract

Biochemical studies in Azoarcus sp. strain T have demonstrated that anaerobic oxidation of both toluene and m-xylene is initiated by addition of the aromatic hydrocarbon to fumarate, forming benzylsuccinate and 3-methyl benzylsuccinate, respectively. Partially purified benzylsuccinate synthase was previously shown to catalyze both of these addition reactions. In this study, we identified and sequenced the genes encoding benzylsuccinate synthase from Azoarcus sp. strain T and examined the role of this enzyme in both anaerobic toluene and m-xylene mineralization. Based on reverse transcription-PCR experiments and transcriptional start site mapping, we found that the structural genes encoding benzylsuccinate synthase, bssCAB, together with two additional genes, bssD and bssE, were organized in an operon in the order bssDCABE. bssD is believed to encode an activating enzyme, similar in function to pyruvate formate-lyase activase. bssE shows homology to tutH from Thauera aromatica strain T1, whose function is currently unknown. A second operon that is upstream of bssDCABE and divergently transcribed contains two genes, tdiS and tdiR. The predicted amino acid sequences show similarity to sensor kinase and response regulator proteins of prokaryotic two-component regulatory systems. A chromosomal null bssA mutant was constructed (the bssA gene encodes the alpha-subunit of benzylsuccinate synthase). This bssA null mutant strain was unable to grow under denitrifying conditions on either toluene or m-xylene, while growth on benzoate was unaffected. The growth phenotype of the DeltabssA mutant could be rescued by reintroducing bssA in trans. These results demonstrate that benzylsuccinate synthase catalyzes the first step in anaerobic mineralization of both toluene and m-xylene.

Abstract

The distribution of aqueous Pb(II) sorbed at the interface between Burkholderia cepacia biofilms and hematite (alpha-Fe(2)O(3)) or corundum (alpha-Al(2)O(3)) surfaces has been probed by using an application of the long-period x-ray standing wave technique. Attached bacteria and adsorbed organic matter may interfere with sorption processes on metal oxide surfaces by changing the characteristics of the electrical double layer at the solid-solution interface, blocking surface sites, or providing a variety of new sites for metal binding. In this work, Pb L(alpha) fluorescence yield profiles for samples equilibrated with 10(-7) to 10(-3.8) M Pb(II) were measured and modeled to determine quantitatively the partitioning of Pb(II) at the biofilm-metal oxide interface. Our data show that the reactive sites on the metal oxide surfaces were not passivated by the formation of a monolayer biofilm. Instead, high-energy surface sites on the metal oxides form the dominant sink for Pb(II) at submicromolar concentrations, following the trend alpha-Fe(2)O(3) (0001) > alpha-Al(2)O(3) (1102) > alpha-Al(2)O(3) (0001), despite the greater site density within the overlying biofilms. At [Pb] > 10(-6) M, significant Pb uptake by the biofilms was observed.

Abstract

The first step in anaerobic ethylbenzene mineralization in denitrifying Azoarcus sp. strain EB1 is the oxidation of ethylbenzene to (S)-(-)-1-phenylethanol. Ethylbenzene dehydrogenase, which catalyzes this reaction, is a unique enzyme in that it mediates the stereoselective hydroxylation of an aromatic hydrocarbon in the absence of molecular oxygen. We purified ethylbenzene dehydrogenase to apparent homogeneity and showed that the enzyme is a heterotrimer (alphabetagamma) with subunit masses of 100 kDa (alpha), 35 kDa (beta), and 25 kDa (gamma). Purified ethylbenzene dehydrogenase contains approximately 0.5 mol of molybdenum, 16 mol of iron, and 15 mol of acid-labile sulfur per mol of holoenzyme, as well as a molydopterin cofactor. In addition to ethylbenzene, purified ethylbenzene dehydrogenase was found to oxidize 4-fluoro-ethylbenzene and the nonaromatic hydrocarbons 3-methyl-2-pentene and ethylidenecyclohexane. Sequencing of the encoding genes revealed that ebdA encodes the alpha subunit, a 974-amino-acid polypeptide containing a molybdopterin-binding domain. The ebdB gene encodes the beta subunit, a 352-amino-acid polypeptide with several 4Fe-4S binding domains. The ebdC gene encodes the gamma subunit, a 214-amino-acid polypeptide that is a potential membrane anchor subunit. Sequence analysis and biochemical data suggest that ethylbenzene dehydrogenase is a novel member of the dimethyl sulfoxide reductase family of molybdopterin-containing enzymes.

Abstract

The novel enzyme benzylsuccinate synthase initiates anaerobic toluene metabolism by catalyzing the addition of toluene to fumarate, forming benzylsuccinate. Based primarily on its sequence similarity to the glycyl radical enzymes, pyruvate formate-lyase and anaerobic ribonucleotide reductase, benzylsuccinate synthase was speculated to be a glycyl radical enzyme. In this report we use EPR spectroscopy to demonstrate for the first time that active benzylsuccinate synthase from the denitrifying bacterium Azoarcus sp. strain T harbors an oxygen-sensitive stable organic free radical. The EPR signal of the radical was centered at g = 2.0021 and was characterized by a major 2-fold splitting of about 1.5 millitesla. The strong similarities between the EPR signal of the benzylsuccinate synthase radical and that of the glycyl radicals of pyruvate formate-lyase and anaerobic ribonucleotide reductase provide evidence that the benzylsuccinate synthase radical is located on a glycine residue, presumably glycine 828 in Azoarcus sp. strain T benzylsuccinate synthase.

Abstract

Aromatic and aliphatic hydrocarbons are the main constituents of petroleum and its refined products. Whereas degradation of hydrocarbons by oxygen-respiring microorganisms has been known for about a century, utilization of hydrocarbons under anoxic conditions has been investigated only during the past decade. Diverse strains of anaerobic bacteria have been isolated that degrade toluene anaerobically, using nitrate, iron(III), or sulfate as electron acceptors. Also, other alkylbenzenes such as m-xylene or ethylbenzene are utilized by a number of strains. The capacity for anaerobic utilization of alkylbenzenes has been observed in members of the alpha-, beta-, gamma- and delta-subclasses of the Proteobacteria. Furthermore, denitrifying bacteria and sulfate-reducing bacteria with the capacity for anaerobic alkane degradation have been isolated, which are members of the beta- and delta-subclass, respectively. The mechanism of the activation of hydrocarbons as apolar molecules in the absence of oxygen is of particular interest. The biochemistry of anaerobic toluene degradation has been studied in detail. Toluene is activated by addition to fumarate to yield benzylsuccinate, which is then further metabolized via benzoyl-CoA. The toluene-activating enzyme presents a novel type of glycine radical protein. Another principle of anaerobic alkylbenzene activation has been observed in the anaerobic degradation of ethylbenzene. Ethylbenzene in denitrifying bacteria is dehydrogenated to 1-phenylethanol and further to acetophenone; the latter is also metabolized to benzoyl-CoA. Naphthalene is presumably activated under anoxic conditions by a carboxylation reaction. Investigations into the pathway of anaerobic alkane degradation are only at the beginning. The saturated hydrocarbons are most likely activated by addition of a carbon compound rather than by desaturation and hydration, as speculated about in some early studies. An anaerobic oxidation of methane with sulfate as electron acceptor has been documented in aquatic sediments. The process is assumed to involve a reversal of methanogenesis catalyzed by Archaea, and scavenge of an electron-carrying metabolite by sulfate-reducing bacteria. Among unsaturated non-aromatic hydrocarbons, anaerobic bacterial degradation has been demonstrated and investigated with n-alkenes, alkenoic terpenes and the alkyne, acetylene.

Abstract

The initial enzymatic steps in anaerobic m-xylene oxidation were studied in Azoarcus sp. strain T, a denitrifying bacterium capable of mineralizing m-xylene via 3-methylbenzoate. Permeabilized cells of m-xylene-grown Azoarcus sp. strain T catalyzed the addition of m-xylene to fumarate to form (3-methylbenzyl)succinate. In the presence of succinyl coenzyme A (CoA) and nitrate, (3-methylbenzyl)succinate was oxidized to E-(3-methylphenyl)itaconate (or a closely related isomer) and 3-methylbenzoate. Kinetic studies conducted with permeabilized cells and whole-cell suspensions of m-xylene-grown Azoarcus sp. strain T demonstrated that the specific rate of in vitro (3-methylbenzyl)succinate formation accounts for at least 15% of the specific rate of in vivo m-xylene consumption. Based on these findings, we propose that Azoarcus sp. strain T anaerobically oxidizes m-xylene to 3-methylbenzoate (or its CoA thioester) via (3-methylbenzyl)succinate and E-(3-methylphenyl)itaconate (or its CoA thioester) in a series of reactions that are analogous to those recently proposed for anaerobic toluene oxidation to benzoyl-CoA. A deuterium kinetic isotope effect was observed in the (3-methylbenzyl)succinate synthase reaction (and the benzylsuccinate synthase reaction), suggesting that a rate-determining step in this novel fumarate addition reaction involves breaking a C-H bond.

Abstract

Gliding motility is observed in a large variety of phylogenetically unrelated bacteria. Gliding provides a means for microbes to travel in environments with a low water content, such as might be found in biofilms, microbial mats, and soil. Gliding is defined as the movement of a cell on a surface in the direction of the long axis of the cell. Because this definition is operational and not mechanistic, the underlying molecular motor(s) may be quite different in diverse microbes. In fact, studies on the gliding bacterium Myxococcus xanthus suggest that two independent gliding machineries, encoded by two multigene systems, operate in this microorganism. One machinery, which allows individual cells to glide on a surface, independent of whether the cells are moving alone or in groups, requires the function of the genes of the A-motility system. More than 37 A-motility genes are known to be required for this form of movement. Depending on an additional phenotype, these genes are divided into two subclasses, the agl and cgl genes. Videomicroscopic studies on gliding movement, as well as ultrastructural observations of two myxobacteria, suggest that the A-system motor may consist of multiple single motor elements that are arrayed along the entire cell body. Each motor element is proposed to be localized to the periplasmic space and to be anchored to the peptidoglycan layer. The force to glide which may be generated here is coupled to adhesion sites that move freely in the outer membrane. These adhesion sites provide a specific contact with the substratum. Based on single-cell observations, similar models have been proposed to operate in the unrelated gliding bacteria Flavobacterium johnsoniae (formerly Cytophaga johnsonae), Cytophaga strain U67, and Flexibacter polymorphus (a filamentous glider). Although this model has not been verified experimentally, M. xanthus seems to be the ideal organism with which to test it, given the genetic tools available. The second gliding motor in M. xanthus controls cell movement in groups (S-motility system). It is dependent on functional type IV pili and is operative only when cells are in close proximity to each other. Type IV pili are known to be involved in another mode of bacterial surface translocation, called twitching motility. S-motility may well represent a variation of twitching motility in M. xanthus. However, twitching differs from gliding since it involves cell movements that are jerky and abrupt and that lack the organization and smoothness observed in gliding. Components of this motor are encoded by genes of the S-system, which appear to be homologs of genes involved in the biosynthesis, assembly, and function of type IV pili in Pseudomonas aeruginosa and Neisseria gonorrhoeae. How type IV pili generate force in S-motility is currently unknown, but it is to be expected that ongoing physiological, genetic, and biochemical studies in M. xanthus, in conjunction with studies on twitching in P. aeruginosa and N. gonorrhoeae, will provide important insights into this microbial motor. The two motility systems of M. xanthus are affected to different degrees by the MglA protein, which shows similarity to a small GTPase. Bacterial chemotaxis-like sensory transduction systems control gliding motility in M. xanthus. The frz genes appear to regulate gliding movement of individual cells and movement by the S-motility system, suggesting that the two motors found in this bacterium can be regulated to result in coordinated multicellular movements. In contrast, the dif genes affect only S-system-dependent swarming.

Abstract

Benzylsuccinate synthase, which catalyzes the anaerobic addition of the methyl carbon of toluene to fumarate, has recently been reported in several denitrifying and sulfate-reducing, toluene-degrading bacteria. In substrate range studies with partially purified benzylsuccinate synthase from denitrifying Azoarcus sp. strain T, benzylsuccinate analogs were observed as a result of fumarate addition to the following toluene surrogates: xylenes, monofluorotoluenes, benzaldehyde, and 1-methyl-1-cyclohexene (but not 4-methyl-l-cyclohexene or methylcyclohexane). Benzylsuccinate was also observed as a result of toluene addition to maleate, but no products were observed from assays with toluene and either crotonate or trans-glutaconate. Toluene-maleate addition, like toluene-fumarate addition, resulted in highly stereospecific formation of the (+)-benzylsuccinic acid enantiomer [(R)-2-benzyl-3-carboxypropionic acid]. The previously reported finding that the methyl H atom abstracted from toluene is retained in the succinyl moiety of benzylsuccinate was found to apply to several toluene surrogates. The implications of these observations for the mechanism of benzylsuccinate synthase will be discussed.

Abstract

Anaerobic mineralization of ethylbenzene by the denitrifying bacterium Azoarcus sp. strain EB1 was recently shown to be initiated by dehydrogenation of ethylbenzene to 1-phenylethanol. 1-Phenylethanol is converted to benzoate (benzoyl coenzyme A) via acetophenone as transient intermediate. We developed in vitro assays to examine ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities in cell extracts of this strain. With p-benzoquinone as the electron acceptor, cell extracts of Azoarcus sp. strain EB1 catalyzed ethylbenzene oxidation at a specific rate of 10 nmol min(-1) [mg of protein](-1) and an apparent K(m) for ethylbenzene of approximately 60 microM. The membrane-associated ethylbenzene dehydrogenase activity was found to oxidize 4-fluoroethylbenzene and propylbenzene but was unable to transform 4-chloro-ethylbenzene, the ethyltoluenes, and styrene. Enzymatic ethylbenzene oxidation was stereospecific, with (S)-(-)-1-phenylethanol being the only enantiomer detected by chiral high-pressure liquid chromatography analysis. Moreover, cell extracts catalyzed the oxidation of (S)-(-)-1-phenylethanol but not of (R)-(+)-1-phenylethanol to acetophenone. When cell extracts were dialyzed, (S)-(-)-1-phenylethanol oxidation occurred only in the presence of NAD(+), suggesting that NAD(+) is the physiological electron acceptor of 1-phenylethanol dehydrogenase. Both ethylbenzene dehydrogenase and 1-phenylethanol dehydrogenase activities were present in Azoarcus sp. strain EB1 cells that were grown anaerobically on ethylbenzene, 1-phenylethanol, and acetophenone, but these activities were absent in benzoate-grown cells.

A chimeric prokaryotic ancestry of mitochondria and primitive eukaryotesPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAKarlin, S., Brocchieri, L., Mrazek, J., Campbell, A. M., Spormann, A. M.1999; 96 (16): 9190-9195

Abstract

We provide data and analysis to support the hypothesis that the ancestor of animal mitochondria (Mt) and many primitive amitochondrial (a-Mt) eukaryotes was a fusion microbe composed of a Clostridium-like eubacterium and a Sulfolobus-like archaebacterium. The analysis is based on several observations: (i) The genome signatures (dinucleotide relative abundance values) of Clostridium and Sulfolobus are compatible (sufficiently similar) and each has significantly more similarity in genome signatures with animal Mt sequences than do all other available prokaryotes. That stable fusions may require compatibility in genome signatures is suggested by the compatibility of plasmids and hosts. (ii) The expanded energy metabolism of the fusion organism was strongly selective for cementing such a fusion. (iii) The molecular apparatus of endospore formation in Clostridium serves as raw material for the development of the nucleus and cytoplasm of the eukaryotic cell.

Abstract

Gliding movements of individual isolated Myxococcus xanthus cells depend on the genes of the A-motility system (agl and cgl genes). Mutants carrying defects in those genes are unable to translocate as isolated cells on solid surfaces. The motility defect of cgl mutants can be transiently restored to wild type by extracellular complementation upon mixing mutant cells with wild-type or other motility mutant cells. To develop a molecular understanding of the function of a Cgl protein in gliding motility, we cloned the cglB wild-type allele by genetic complementation of the mutant phenotype. The nucleotide sequence of a 2.85-kb fragment was determined and shown to encode two complete open reading frames. The CglB protein was determined to be a 416-amino-acid putative lipoprotein with an unusually high cysteine content. The CglB antigen localized to the membrane fraction. The swarming and gliding defects of a constructed DeltacglB mutant were fully restored upon complementation with the cglB wild-type allele. Experiments with a cglB allele encoding a CglB protein with a polyhistidine tag at the C terminus showed that this allele also promoted wild-type levels of swarming and single-cell gliding, but was unable to stimulate DeltacglB cells to move. Possible functions of CglB as a mechanical component or as a signal protein in single cell gliding are discussed.

Abstract

Myxococcus xanthus cells move on a solid surface by gliding motility. Several genes required for gliding motility have been identified, including those of the A- and S-motility systems as well as the mgl and frz genes. However, the cellular defects in gliding movement in many of these mutants were unknown. We conducted quantitative, high-resolution single-cell motility assays and found that mutants defective in mglAB or in cglB, an A-motility gene, reversed the direction of gliding at frequencies which were more than 1 order of magnitude higher than that of wild type cells (2.9 min-1 for DeltamglAB mutants and 2.7 min-1 for cglB mutants, compared to 0.17 min-1 for wild-type cells). The average gliding speed of DeltamglAB mutant cells was 40% of that of wild-type cells (on average 1.9 micrometers/min for DeltamglAB mutants, compared to 4.4 micrometers/min for wild-type cells). The mglA-dependent reversals and gliding speeds were dependent on the level of intracellular MglA protein: mglB mutant cells, which contain only 15 to 20% of the wild-type level of MglA protein, glided with an average reversal frequency of about 1.8 min-1 and an average speed of 2.6 micrometers/min. These values range between those exhibited by wild-type cells and by DeltamglAB mutant cells. Epistasis analysis of frz mutants, which are defective in aggregation and in single-cell reversals, showed that a frzD mutation, but not a frzE mutation, partially suppressed the mglA phenotype. In contrast to mgl mutants, cglB mutant cells were able to move with wild-type speeds only when in close proximity to each other. However, under those conditions, these mutant cells were found to glide less often with those speeds. By analyzing double mutants, the high reversing movements and gliding speeds of cglB cells were found to be strictly dependent on type IV pili, encoded by S-motility genes, whereas the high-reversal pattern of mglAB cells was only partially reduced by a pilR mutation. These results suggest that the MglA protein is required for both control of reversal frequency and gliding speed and that in the absence of A motility, type IV pilus-dependent cell movement includes reversals at high frequency. Furthermore, mglAB mutants behave as if they were severely defective in A motility but only partially defective in S motility.

Abstract

Recent studies of anaerobic toluene catabolism have demonstrated a novel reaction for anaerobic hydrocarbon activation: the addition of the methyl carbon of toluene to fumarate to form benzylsuccinate. In vitro studies of the anaerobic benzylsuccinate synthase reaction indicate that the H atom abstracted from the toluene methyl group during addition to fumarate is retained in the succinyl moiety of benzylsuccinate. Based on structural studies of benzylsuccinate formed during anaerobic, in vitro assays with denitrifying, toluene-mineralizing strain T, we now report the following characteristics of the benzylsuccinate synthase reaction: (i) it is highly stereospecific, resulting in >95% formation of the (+)-benzylsuccinic acid enantiomer [(R)-2-benzyl-3-carboxypropionic acid], and (ii) active benzylsuccinate synthase does not contain an abstracted methyl H atom from toluene at the beginning or at the end of a catalytic cycle.

Abstract

Reductive dehalogenation of vinyl chloride (VC) was studied in an anaerobic mixed bacterial culture. In growth experiments, ethene formation from VC increased exponentially at a rate of about 0.019 h(sup-1). Reductive VC dehalogenation was measured in vitro by using cell extracts of the mixed culture. The apparent K(infm) for VC was determined to be about 76 (mu)M; the V(infmax) was about 28 nmol (middot) min(sup-1) (middot) mg of protein(sup-1). The VC-dehalogenating activity was membrane associated. Propyl iodide had an inhibitory effect on the VC-dehalogenating activity in the in vitro assay. However, this inhibition could not be reversed by illumination. Cell extracts also catalyzed the reductive dehalogenation of cis-1,2-dichloroethene (cis-DCE) and, at a lower rate, of trichloroethene (TCE). Tetrachloroethene (PCE) was not transformed. The results indicate that the reductive dehalogenation of VC and cis-DCE described here is different from previously reported reductive dehalogenation of PCE and TCE.

Abstract

Permeabilized cells of toluene-mineralizing, sulfate-reducing strain PRTOL1 catalyzed the addition of toluene to fumarate to form benzylsuccinate under anaerobic conditions. Recent in vitro studies with two toluene-mineralizing, denitrifying bacteria demonstrated the same fumarate addition reaction and indicated that it may be the first step of anaerobic toluene degradation. This study with strain PRTOL1 shows that anaerobic toluene activation by fumarate addition occurs in bacteria as disparate as sulfate-reducing and denitrifying species (members of the delta and beta subclasses of the Proteobacteria, respectively).

Abstract

Anaerobic assays conducted with strain T, a denitrifying bacterium capable of mineralizing toluene to carbon dioxide, demonstrated that toluene-grown, permeabilized cells catalyzed the addition of toluene to fumarate to form benzylsuccinate. This reaction was not dependent on the presence of coenzyme A (CoA) or ATP. In the presence of CoA, formation of E-phenylitaconate from benzylsuccinate was also observed. Kinetic studies demonstrated that the specific rate of benzylsuccinate formation from toluene and fumarate in assays with permeabilized cells was >30% of the specific rate of toluene consumption in whole-cell suspensions with nitrate; this observation suggests that benzylsuccinate formation may be the first reaction in anaerobic toluene degradation by strain T. Use of deuterium-labeled toluene and gas chromatography-mass spectrometry indicated that the H atom abstracted from the toluene methyl group during addition to fumarate was retained in the succinyl moiety of benzylsuccinate. In this study, no evidence was found to support previously proposed reactions of toluene with acetyl-CoA or succinyl-CoA. Toluene-grown, permeabilized cells of strain T also catalyzed the addition of o-xylene to fumarate to form (2-methylbenzyl)succinate. o-Xylene is not a growth substrate for strain T, and its transformation was probably cometabolic. With the exception of specific reaction rates, the observed characteristics of the toluene-fumarate addition reaction (i.e., retention of a methyl H atom and independence from CoA and ATP) also apply to the o-xylene-fumarate addition reaction. Thus, addition to fumarate may be a biochemical strategy to anaerobically activate a range of methylbenzenes.

Abstract

Initial reactions in anaerobic oxidation of ethylbenzene were investigated in a denitrifying bacterium, strain EB1. Cells of strain EB1 mineralized ethylbenzene to CO2 under denitrifying conditions, as demonstrated by conversion of 69% of [14C]ethylbenzene to 14CO2. In anaerobic suspensions of strain EB1 cells metabolizing ethylbenzene, the transient formation and consumption of 1-phenylethanol, acetophenone, and an as yet unidentified compound were observed. On the basis of growth experiments and spectroscopic data, the unknown compound is proposed to be benzoyl acetate. Cell suspension experiments using H2(18)O demonstrated that the hydroxyl group of the first product of anoxic ethylbenzene oxidation, 1-phenylethanol, is derived from water. A tentative pathway for anaerobic ethylbenzene mineralization by strain EB1 is proposed.

Abstract

A novel sulfate-reducing bacterium isolated from fuel-contaminated subsurface soil, strain PRTOL1, mineralizes toluene as the sole electron donor and carbon source under strictly anaerobic conditions. The mineralization of 80% of toluene carbon to CO2 was demonstrated in experiments with [ring-U-14C]toluene; 15% of toluene carbon was converted to biomass and nonvolatile metabolic by-products, primarily the former. The observed stoichiometric ratio of moles of sulfate consumed per mole of toluene consumed was consistent with the theoretical ratio for mineralization of toluene coupled with the reduction of sulfate to hydrogen sulfide. Strain PRTOL1 also transforms o- and p-xylene to metabolic products when grown with toluene. However, xylene transformation by PRTOL1 is slow relative to toluene degradation and cannot be sustained over time. Stable isotope-labeled substrates were used in conjunction with gas chromatography-mass spectrometry to investigate the by-products of toluene and xylene metabolism. The predominant by-products from toluene, o-xylene, and p-xylene were benzylsuccinic acid, (2-methylbenzyl)succinic acid, and 4-methylbenzoic acid (or p-toluic acid), respectively. Metabolic by-products accounted for nearly all of the o-xylene consumed. Enzyme assays indicated that acetyl coenzyme A oxidation proceeded via the carbon monoxide dehydrogenase pathway. Compared with the only other reported toluene-degrading, sulfate-reducing bacterium, strain PRTOL1 is distinct in that it has a novel 16S rRNA gene sequence and was derived from a freshwater rather than marine environment.

Abstract

Prokaryotic gliding motility is described as the movement of a cell on a solid surface in the direction of the cell's long axis, but its mechanics are unknown. To investigate the basis of gliding, movements of individual Myxococcus xanthus cells were monitored by employing a video microscopy method by which displacements as small as 0.03 micron could be detected and speeds as low as 1 micron/min could be resolved. Single cells were observed to glide with speeds varying between 1 and 20 microns/min. We found that speed variation was due to differences in distance between the moving cell and the nearest cell. Cells separated by less than one cell diameter (0.5 micron) moved with an average speed of 5.0 micron/min, whereas cells separated by more than 0.5 micron glided with an average speed of 3.8 microns/min. The power to glide was found to be carried separately at both ends of a cell.